ABSTRACT: KDM5 histone lysine demethylases as potential novel myeloid tumor suppressors. Mutations in Isocitrate Dehydrogenase (IDH1 and IDH2) are present in over 20% of cases of de novo normal karyotype AML and in 10-20% of cases of secondary AML that result from leukemic transformation of myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN). Mutant IDH transforms cells by producing R-2-hydroxyglutarate (R-2HG), an oncometabolite that can inhibit the activity of a number of cellular enzymes, including TET2, a myeloid tumor suppressor that regulates the methylation state of DNA. It is not known if R-2HG has other pathogenic targets besides TET2 in AML. However, the phenotypes of IDH mutant and TET2 mutant myeloid diseases are quite different. This observation forms the basis of the premise of our proposal, which is that inhibition of other pathways by R-2HG contributes to mutant IDH-mediated transformation. We performed an unbiased positive-selection CRISPR-Cas9 screen to identify novel tumor suppressors in AML, and we identified two histone lysine demethylases, KDM5A and KDM5C, as potential pathogenic targets of R-2HG in IDH mutant AML. Our central hypothesis is that KDM5A and KDM5C regulate hematopoietic stem cell function, and that disruption of KDM5A and KDM5C activity by R-2HG contributes to mutant IDH-mediated transformation. Our proposed studies address this hypothesis by asking three key questions. First, what is the mechanism by which KDM5 loss promotes cytokine-independent proliferation of TF-1 cells, an established factor-dependent human AML cell line? cDNA rescue experiments and genomic profiling of histone lysine methylation and transcription will be used, in conjunction with genetic manipulation of KDM5 enzymes, to elucidate the unique and shared functions of KDM5 isoforms in AML. Second, what evidence is there that KDM5 enzymes are inhibited by R-2HG in IDH mutant AML? We will profile the histone methylation state of primary IDH mutant and IDH wild-type AML patient samples, and will characterize the epigenetic and transcriptional states of isogenic cell lines that express wild-type and mutant IDH to determine if inhibition of KDM5 enzymes by R-2HG contributes to the mutant IDH-mediated transformation. And finally, how does loss of Kdm5a affect normal murine hematopoiesis and clonal hematopoiesis induced by loss of canonical myeloid tumor suppressors? We will employ conditional Kdm5a knock-out mice to perform detailed analyses of hematopoiesis in mice that lack Kdm5a alone and that lack Kdm5a in combination with Dnmt3a, Nf1 or Tet2. The answers to these questions will give us a greater understanding of the role of KDM5 demethylases in normal and malignant hematopoiesis, and in IDH mutant AML in particular. This work is conceptually innovative in that it will establish a novel epigenetic mechanism that contributes to AML, and is significant in that it has the potential to lead to novel therapeutic approaches to treat patients with leukemia.